Chrodrimanins K–N and Related Meroterpenoids from the Fungus

Article pubs.acs.org/jnp

Chrodrimanins K−N and Related Meroterpenoids from the Fungus Penicillium sp. SCS-KFD09 Isolated from a Marine Worm, Sipunculus nudus Fan-Dong Kong,† Qing-Yun Ma,† Sheng-Zhuo Huang,† Pei Wang,† Jun-Feng Wang,‡ Li-Man Zhou,† Jing-Zhe Yuan,† Hao-Fu Dai,*,† and You-Xing Zhao*,† †

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou 571101, People’s Republic of China ‡ CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Matria Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China S Supporting Information *

ABSTRACT: Six new meroterpenoids, chrodrimanins K−N (1− 4), including two uncommon chlorinated ones (1 and 2), and verruculides B2 (5) and B3 (6), as well as seven known ones (7− 13), were isolated from the fermentation broth of Penicillium sp. SCS-KFD09 isolated from a marine worm, Sipunculus nudus, from Haikou Bay, China. The structures including the absolute configurations of the new compounds were unambiguously elucidated by spectroscopic data, X-ray diffraction analysis, and ECD spectra analysis along with quantum ECD calculations. In addition, the X-ray crystal structures and absolute configurations of two previously reported meroterpenoids, chrodrimanins F (9) and A (11), are described for the first time. Compounds 1, 4, and 7 displayed anti-H1N1 activity with IC50 values of 74, 58, and 34 μM, respectively, while compound 5 showed weak inhibitory activity against Staphylococcus aureus with an MIC of 32 μg/mL.

M

Bay, China. Subsequent chemical investigation on the EtOAc extract of the fermentation broth led to the identification of six new meroterpenoids, chrodrimanins K−N (1−4), including two uncommon chlorinated ones (1 and 2), and verruculides B2 (5) and B3 (6), along with seven known ones: 3hydroxypentacecilide A (7),6 chrodrimanin H (8),3 chrodrimanin F (9),3 chrodrimanin E (10),3 chrodrimanin A (11),2 chrodrimanin B (12),2 and verruculide B (13).7 Herein, the isolation, structure elucidation, and bioactivities of these compounds are described.

eroterpenoids are a class of natural products of mixed biosynthetic origin, with a terpenoid as one of the structural components.1 Chrodrimanins,2−4 pentacecilides,5,6 and verruculide A7 represent a type of meroterpenoid constructed from a drimane-type sesquiterpene unit and a C10 polyketide unit, the latter of which is cyclized to be a 6,8dihydroxy-3-methylisochroman-1-one or a 3,6,8-trihydroxy-3,4dihydronaphthalen-1-one moiety. Only 15 analogues have been reported, including chrodrimanins A−J,2−4 pentacecilides A− D,5,6 and verruculide A.7 All of these compounds are fungal metabolites and share an interestingly 6/6/6/6/6 pentacyclic ring framework. In addition, verruculide B, a derivative considered to be the biosynthetic precursor of these compounds, was also coisolated with verruculide A.7 These compounds showed various bioactivities, including insecticidal activity,2,3 lipid droplet formation inhibition,6 protein tyrosine phosphatase 1B inhibitory activity,7 and antiviral activity.4 Marine fungi have long been an important source of structurally new and biologically active metabolites, many of which have been used as therapeutic drug leads.8 During our ongoing search for new bioactive metabolites of marine fungal origin,9 the fungus Penicillium sp. SCS-KFD09 was isolated and identified from a marine worm, Sipunculus nudus, from Haikou © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals. The molecular formula was determined as C25H31ClO6 by HRESIMS, containing an additional Cl substituent in comparison with the formerly described chrodrimanin H (8).3 The 1H, 13C, and HSQC NMR spectra of 1 showed signals for one ketone carbonyl at δC 217.4, one ester carbonyl at δC 170.0, one fully substituted benzene ring (δC 110.5, 136.5, 102.4, 158.1, 108.1, and 154.2), five endocyclic methylenes, four sp3 methines Received: November 17, 2016 Published: February 17, 2017 1039

DOI: 10.1021/acs.jnatprod.6b01061 J. Nat. Prod. 2017, 80, 1039−1047

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and H-2 and HMBC correlations from H2-1 and H-2 to C-3 (Figure 1). The coupling constants (Table 1) and ROESY correlations of 2 resembled those of 1 (Figure 2), except for an additional ROESY correlation observed between H-2 and H-5, which indicated they were both α-oriented. Finally, the absolute configuration of 2 was determined by a single-crystal X-ray diffraction experiment (Figure 3). The final refinement of the Cu Kα data resulted in a 0.042(11) Flack parameter, allowing for the unambiguous assignment of the absolute configuration as 2S, 5R, 7S, 8R, 9S, 10S, and 8′R. Therefore, compound 2, named chrodrimanin L, was assigned. Compound 3 was isolated as colorless crystals. Its molecular formula was established as C25H34O7 by HRESIMS, bearing one more oxygen atom compared to chrodrimanin F (9).3 Comparison of the 1H and 13C NMR data of 3 with those of 9 revealed the presence of one additional oxygenated methine (δC/H 72.7/3.96) and the disappearance of the C-1 methylene, which implied the oxygenation of this methylene to a hydroxylated methine group at C-1 in 3. This was corroborated by sequential COSY correlations of one oxymethine proton H1 to another oxymethine proton H-3 (δH 3.76) through H2-2 (δH 1.83, 1.99), as well as an HMBC correlation from the singlet methyl protons H3-15 (δH 1.14) to the oxymethine carbon C-1 (Figure 1). The remaining substructure of 3 was found to be identical to that of 9, as confirmed by detailed interpretation of the 2D NMR data (Figure 1). Hence, compound 3 was identified as 1-hydroxychrodrimanin F. The J values and ROESY relationships of 3 were similar to those of 1 (Figure 2), except for the additional ROESY correlations of H-3/H-5 and H-1/H3-15, which determined the β- and αorientations of H-1 and H-3, respectively. This assignment was further confirmed by X-ray single-crystal diffraction analysis (Figure 3). From a biosynthetic consideration, the absolute configurations at the C-5, C-7, C-8, C-9, C-10, and C-8′ positions of 3 were deduced be identical to those of 1 and 2. Finally, the absolute configuration of 3 was assigned as 1S, 3S, 5S, 7S, 8R, 9S, 10S, and 8′R by comparison of its experimental electronic circular dichroism (ECD) curve with those of 1 and 2, which showed close similarity (Figure 4). Compound 4 was isolated as colorless crystals. It exhibited a prominent sodium adduct ion peak at m/z 511.2301 [M + Na]+ in the HRESIMS spectrum, suggesting a molecular formula of C27H36O8, with an additional acetoxy group compared to 9. Their NMR data were also quite similar, except for the presence of additional signals for acetoxy (δC/H 170.7, 20.8/ 2.14) and oxygenated methine (δC/H 64.3/6.12) groups, as well as the absence of signals for CH2-7′ in 4. These data led to a deduction that one acetoxy group was located at C-7′ compared to 9, as confirmed by COSY correlations of H-8′ with H3-9′ (δH 1.46) and the oxymethine proton H-7′ and an HMBC correlation from H-7′ to the acetoxy carbonyl carbon (Figure 1). Comprehensive analysis of the 1D and 2D NMR spectra revealed the remaining structure of 4 to be identical to that of 9. Consequently, compound 4 was determined to be 7′acetoxychrodrimanin F. The relative configuration of the stereogenic centers C-3, C-5, C-7, C-8, C-9, and C-10 in the sesquiterpene skeleton of 4 was assigned to be the same as those of 9 according to the similar J values (Table 1) and ROESY data (Figure 2). However, no diagnostic ROESY crosspeaks could be used to correlate the stereogenic centers C-7′ and C-8′ with the other stereogenic centers in the sesquiterpene skeleton. In order to complete the relative configuration assignment, 4 was crystallized and, by use of X-

including two oxygenated (δC/H 73.3/4.11 and 75.0/4.64), three nonprotonated carbons (δC 47.0, 35.9, and 79.7), and five methyls (Table 1). Detailed comparison of these data with 8 indicated the presence of the same drimane-type sesquiterpenoid substructure in 1, as evidenced by COSY correlations of H2-1/H2-2, H-5/H2-6/H-7, and H-9/H2-11 and by HMBC correlations from H3-13 (δH 1.11) and H3-14 (δH 1.12) to C-3, C-4, and C-5 (δC 43.8), from H3-15 (δH 1.03) to C-1 (δC 32.0), C-5, C-9 (δC 40.9), and C-10 (δC 35.9), and from H3-12 (δH 1.41) to C-7, C-8, and C-9 (Figure 1). The other unassigned signals for the isochromanone moiety also resembled those of 8, except for the disappearance of the singlet H-5′ proton signal. Furthermore, HMBC correlations from H2-7′ (δH 2.85, 2.73) to aromatic nonprotonated carbons C-1′, C-2′, and C-3′, from H2-11 (δH 2.55, 2.50) to aromatic nonprotonated carbons C-1′, C-2′, and C-6′, from the oxymethine proton H-8′ to the conjugated ester carbony C-10′, and from the hydrogenbonded hydroxy 4′-OH proton (δH 11.70) to C-3′, C-4′, and C-5′ and COSY correlations of H3-9/H-8/H2-7 were observed (Figure 1). The above data, with the aid of a molecular formula that showed the presence of a Cl atom, assigned 1 as 5′chlorochrodrimanin H. The ROESY correlations of H-5 (δH 1.96) with H3-14 (δH 1.12), H3-12, and H-7 determined these protons to be on the same face of the ring system, whereas correlations of H-6β (δH 2.19) with H3-15 (δH 1.03), H3-15 with H-9 (δH 2.05) and H-11β (δH 2.50), and H-11β with H7′β (δH 2.73) suggested that these protons were on the opposite face of the molecule (Figure 2). H-8′ (δH 4.64) was assigned as α-oriented due to its large coupling constant (J = 12.0 Hz) with H-7′β. To support the above deduction and determine the absolute configuration of 1, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Kα radiation (Figure 3), allowing an explicit assignment of the absolute structure as 5R, 7S, 8R, 9S, 10S, and 8′R on the basis of the Flack parameter of 0.06(3). Thus, compound 1 was elucidated and named chrodrimanin K. Compound 2 was isolated as colorless crystals, whose molecular formula was established as C25H30Cl2O6 by HRESIMS, bearing one more Cl substituent than 1. Comparison of the 1D NMR data between compounds 2 and 1 indicated that they shared the same basic skeleton, with the main differences being the replacement of CH2-2 in 1 by one heteroatom-substituted sp3 methine in 2. This suggested 2 to be 2,5′-dichlorochrodrimanin H based on the analysis of the molecular formula and the characteristic chemical shifts for CH-2 (δC/H 58.1/4.89). The assignment was further corroborated by observation of the COSY correlation between H2-1 1040

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33.6, CH2

217.4, 47.0, 43.8, 28.3,

73.3, 79.7, 40.9, 35.9, 19.8,

23.6, 20.1, 28.9, 22.9, 110.5, 136.5, 102.4, 158.1, 108.1, 154.2, 31.6,

75.0, CH 21.1, CH3 170.0, C

2

3 4 5 6

7 8 9 10 11

12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′

8′ 9′ 10′ CH3CO CH3CO 4′-OH

CH3 CH3 CH3 CH3 C C C C C C CH2

CH C CH C CH2

C C CH CH2

δC, type

32.0, CH2

position

1

δH (J in Hz)

dd (14.2, 2.3) ddd (13.8, 8.4, 2.3) overlap dd (8.4, 7.7)

m ddd (13.0, 9.4, 4.9) m m

1041

dd (16.6, 3.4) dd (16.6, 12.0) m d (6.3)

2.85, 2.73, 4.64, 1.57,

11.70, s

dd (15.9, 13.7) overlap s s s s

2.55, 2.50, 1.41, 1.11, 1.12, 1.03,

2.05, dd (13.7, 5.2)

1.96, 2.19, 1.60, 4.11,

2.02, 1.70, 2.65, 2.45,

1 δC, type

CH3 CH3 CH3 C C C C C C C CH2

CH C CH C CH2

C C CH CH2

75.1, CH 21.1, CH3 169.9, C

23.8, 19.8, 29.7, 24.0, 109.9, 136.5, 102.6, 158.2, 108.2, 153.9, 31.7,

73.2, 79.1, 40.5, 36.7, 19.5,

208.6, 47.0, 42.8, 28.2,

58.1, CH

45.4, CH2

δH (J in Hz)

dd (14.2, 2.3) ddd (14.2, 8.1, 2.1) overlap dd (8.1, 8.1)

dd (16.5, 3.3) dd (16.5, 12.2) m overlap

dd (15.8, 13.1) dd (15.8, 5.1) s s s s

11.76, s

2.88, 2.73, 4.65, 1.56,

2.56, 2.47, 1.46, 1.21, 1.18, 1.00,

1.98, dd (13.0, 5.2)

2.08, 2.20, 1.55, 4.11,

2.97, dd (12.6, 11.7) 1.79, dd (12.6, 7.0) 4.89, dd (11.7, 7.0)

2

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 1−4 in CDCl3 δC, type

CH3 CH3 CH3 CH3 C C C C CH C CH2

CH C CH C CH2

CH C CH CH2

74.9, CH 21.1, CH3 170.2, C

22.7, 15.6, 29.0, 24.6, 111.6, 138.9, 102.0, 162.2, 103.4, 159.3, 31.9,

73.4, 79.5, 43.5, 41.2, 19.4,

73.9, 39.4, 36.8, 26.4,

37.4, CH2

72.7, CH

δH (J in Hz)

dd (14.0, 3.3) ddd (14.0, 8.9, 3.3) overlap dd (8.9, 5.1)

dd (14.7, 13.5) dd (14.7, 5.0) s s s s

dd (16.5, 3.3) dd (16.5, 12.1) m d (6.3)

11.00, s

2.86, 2.69, 4.63, 1.54,

6.31, s

3.14, 2.49, 1.36, 0.85, 1.05, 1.14,

2.11, dd (13.5, 5.0)

1.93, 2.29, 1.64, 4.01,

1.83, ddd (14.1, 4.4, 3.9) 1.99, m 3.76, dd (12.0, 5.1)

3.96, br s

3 δC, type

76.2, 16.6, 169.1, 170.7, 20.8,

20.8, 15.8, 28.9, 24.0, 113.2, 135.8, 102.2, 162.3, 106.2, 159.9, 64.3,

72.9, 80.0, 42.7, 36.1, 19.5,

79.0, 39.4, 44.3, 26.7,

CH CH3 C C CH3

CH3 CH3 CH3 CH3 C C C C CH C CH

CH C CH C CH2

CH C CH CH2

28.4, CH2

34.0, CH2

δH (J in Hz)

dd (13.7, 3.8) ddd (13.7, 9.0, 3.8) m dd (9.0, 4.0)

m overlap m m dd (11.7, 4.8)

overlap overlap s s s s

2.14, s 11.02, s

4.69, qd (6.7, 1.7) 1.46, d (6.7)

6.12, d (1.7)

6.47, s

2.60, 2.60, 2.14, 0.86, 1.00, 1.13,

2.06, dd (12.8, 6.1)

1.31, 2.23, 1.64, 4.06,

1.55, 1.46, 1.79, 1.68, 3.22,

4

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methyls, six sp3 methylenes, three sp3 oxygenated methines, two sp3 oxygenated nonprotonated carbons, two sp2 methines, six sp2 nonprotonated carbons, and one carbonyl carbon by the analysis of HMQC and DEPT spectra. The presence of the 3,4dihydroisocoumarin moiety (C-1′−C-10′) in 5 was deduced by comparison of the related NMR signals with those of verruculide B (13).7 This was further confirmed by sequential COSY data (C-7′−C-9′) and HMBC correlations from H-5′ (δH 6.27) to C-1′ (δC 119.3), C-3′ (δC 101.2), C-4′ (δC 163.8), and C-6′ (δC 164.3) and from H2-7′ (δH 3.03, 2.69) to C-1′, C2′ (δC 140.7), and C-3′, as shown in Figure 2. The remaining signals corresponding to a linear sesquiterpene moiety were also quite similar to those of 13, with the main differences being the replacement of signals for the Δ6 double bond by those for one oxygenated methine (δC/H 77.2/3.40) and one oxygenated nonprotonated carbon (δC 87.2). Analysis of the COSY spectrum allowed the establishment of three isolated spin systems: −CH2 (1)−CH (2), −CH2 (4)−CH2 (5)−CH (6), and −CH2 (8)−CH2 (9)−CH (10) (Figure 1). These moieties were connected by analysis of the HMBC spectrum, from which H3-15 (δH 1.76) to C-2 (δC 124.0), C-3 (δC 136.0), and C-4 (δC 37.4) and H3-14 (δH 1.11) to C-6, C-7, and C-8 (δC 33.3) were observed. The presence of a 2-hydroxypropan-2-yl substituent at C-10 was determined by HMBC correlations from the two singlet methyl protons H3-12 (δH 1.10) and H313 (δH 1.18) to C-10 (δC 85.9) and C-11 (δC 72.7). However, taking the molecular formula into account, one more ring system is required for 5. This could be accounted for by a linkage between C-10 and C-7 through an O atom, which was supported by comparison of NMR data for the same substructure as those of quassiol B,11 kuhistano,12 and (17R,20S,24R)-17,25-dihydroxy-20,24-epoxy-14(18)-malabaricen-3-one.13 Thus, the planar structure of 5 was assigned. Compound 6 was determined to share the same planar structure with 5, due to their same HMBC and COSY data (Figure 1).

Figure 1. Key COSY and HMBC correlations of compounds 1−6.

ray analysis (Figure 3), its relative configuration was fully assigned. The ECD curve of 4 was similar to those of 1 and 2 (Figure 4), suggesting its absolute configuration as 3S, 5R, 7S, 8R, 9S, 10S, 7′R, and 8′R. In order to confirm this deduction, the conformer of 4 obtained from the X-ray analysis was used as the input for the structural optimization by the density functional theory method at the B3LYP/6-31G(d) level in the Gaussian 03 program package.10 The ECD was then calculated by the time-dependent density functional method at the B3LYP/6-31G(d) level. The calculated ECD spectrum of 4 fit well with the measured one (Figure 4), further confirming the above assignment. Compounds 5 and 6 were isolated as colorless oils and assigned the same molecular formula C25H36O7 from HRESIMS peaks at m/z 471.2350 [M + Na]+ and 471.2354 [M + Na]+, requiring eight degrees of unsaturation. The 13C NMR spectrum for each compound showed 25 carbon signals, which were classified into five

Figure 2. Key ROESY correlations of compounds 1−6. 1042

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Figure 3. Molecular plots of compounds 1−4, 9, and 11.

The configurations of the Δ2 double bonds in both compounds 5 and 6 were assigned as E from the ROESY cross-peaks of H-1/H-15 and H-2/H-4 (Figure 2). Further, in the ROESY spectra (Figure 2), correlations of H3-14/H-10 were observed in 5, while correlations of H-6/H-10 were observed in 6, indicating the cis and trans orientation of H-10 and H3-14 in 5 and 6, respectively. Moreover, it has been reported14 that, in the isomers with the two largest substituents of the tetrahydrofuran ring in the trans orientation, a hydrogen corresponding to H-10 appeared as a doublet of doublets, whereas in the cis diastereomers an apparent triplet was observed, which was indeed also observed in the spectra of 5

[δH‑10 3.78, t (7.6)] and 6 [δH‑10 3.70, dd (9.4, 6.2)] (Table1 and 1H NMR spectra in the Supporting Information). Hiroyuki Yamazaki7 et al. have reported the absolute configuration of C8′ in 13 as R by comparison of ECD data ascribed to the isocoumarin moiety with those for (R)-6-hydroxymellein. Analysis of the measured ECD curves for 5 and 6 revealed strong negative Cotton effects around 215, 237, and 273 nm, which were similar to those of 1, 2, and 137 (Figure 4), indicating the R-configuration of C-8′. Compound 7, obtained as a colorless, amorphous powder, was suggested to possess the molecular formula C25H34O5, with one oxygen atom less than 9, on the basis of the ESIMS peak at 1043

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Figure 4. Experimental ECD curves for compounds 1−6, 9, and 11 and calculated ECD curves for 4 and 11.

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m/z 437.2 [M + Na]+. The 1H NMR spectrum (Supporting Information) of 7 was identical with that reported for 3hydroxypentacecilide A,6 which was semisynthesized from the natural precursor pentacecilide A, assigning their same planar structure and relative configuration. On biosynthetic grounds, the absolute configuration of 7 was deduced to be (3S,5R,8S,9S,10S,8′R) in light of the absolute configuration of the co-occurring chrodrimanins, 3 and 4. Thus, compound 7 was assigned as a new natural meroterpenoid and determined to be 3-hydroxypentacecilide A. Compounds 9 and 11 were determined to be chrodrimanins F and A, respectively, by comparison of their 1D NMR data in CDCl3 and [α]25D values (−54 and +13 for 9 and 11, respectively) in MeOH−CHCl3 (1:1) with those reported (−63 and +8 for chrodrimanins F and A, respectively).2,3 In addition, we report the X-ray crystal structures (Figure 3) of 9 and 11 for the first time and originally determined their absolute configurations as (3S,5R,7S,8R,9S,10S,8′R) and (5R,7S,8R,9S,10S,7′R,8′R), respectively, by comparison of experimental ECD data of 9 with those of 1, 2, and 3 and of 11 with that of the calculated spectrum (Figure 4). All of the isolated compounds were evaluated for enzyme inhibitory activity against AChE9 and α-glycosidase,15 antibacterial activity against Staphylococcus aureus,16 and nematicidal activity against Panagrellus redivevus.17 Only compound 5 showed weak inhibitory activity against S. aureus with an MIC of 32 μg/mL (gentamicin sulfate as the positive control, MIC 4.0 μg/mL). The antiviral activities of compounds 1−7 and 13 against influenza A virus (H1N1) were also tested by the CPE inhibition assay,18,19 considering that the anti-H1N1 activities of compounds 8−12 have been reported.4 The results showed that compounds 1, 4, and 7 displayed anti-H1N1 activity with IC50 values of 74, 58, and 34 μM, respectively (ribavirin as positive control, IC50 = 103 μM).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter, and UV spectra were measured on a Beckman DU 640 spectrophotometer. ECD data were collected using a JASCO J-715 spectropolarimeter. IR spectra were taken on a Nicolet Nexus 470 spectrophotometer as KBr discs. NMR spectra were recorded on a Bruker AV-500 spectrometer with tetramethylsilane as an internal standard. ESIMS, HRESIMS, and HREIMS spectra were recorded with a Micromass Autospec-UltimaTOF, API QSTAR Pulsar 1, or Waters Autospec Premier spectrometer. Semipreparative HPLC was carried out using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min) and a π NAP column (Cosmosil-pack, 10 × 250 mm, 5 μm, 4 mL/min). Thinlayer chromatography (TLC) and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao Marine Chemical Factory). The sea salt used was made from the evaporation of seawater collected in Laizhou Bay, China (Weifang HaiHua Yu Feng Chemical Factory). Fungal Material and Fermentation. The fungus SCS-KFD09 was isolated from a marine worm, Sipunculus nudus (HK10404), from Haikou Bay, China, in August 2015. After grinding, the sample (1 g) was diluted to 10−2 g/mL with sterile H2O, 100 μL of which was deposited on a PDA (200 g potato, 20 g glucose, 20 g agar per liter of seawater collected in Haikou Bay, China) plate containing chloramphenicol (100 μg/mL) as a bacterial inhibitor. A single colony was transferred onto another PDA plate and was identified according to its morphological characteristics and 18S rRNA gene sequences (GenBank accession no. KX981597, Supporting Information). A reference culture of Penicillium sp. SCS-KFD09 maintained at −80 °C is deposited in our laboratory. The isolate was cultured on slants of PDA medium at 28 °C for 4 days. Plugs of agar supporting mycelium growth were cut and transferred aseptically to 150 × 1000 mL Erlenmeyer flasks each containing 300 mL of liquid medium (20 g mannitol, 20 g maltose, 10 g glucose, 10 g monosodium glutamate, 3 g yeast extract, 0.5 g corn meal, 0.5 g KH2PO4, 0.3 g MgSO4, 17.5 g Na2HPO4·2H2O, 10.5 g C4H2O7·H2O, 33 g sea salt per liter of tap 1044

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Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 5 and 6 in CD3OD 5

6

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1

24.8, CH2

24.8, CH2

2 3 4

124.0, CH 136.0, C 37.4, CH2

3.25, dd (15.2, 7.1) 3.32, dd (15.2, 7.1) 5.09, t (7.1)

3.26, dd (16.4, 7.1) 3.31, dd (16.4, 7.1) 5.09, t (7.1)

5

31.2, CH2

6 7 8

77.2, CH 87.2, C 33.3, CH2

9

27.5, CH2

position

10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′

85.9, 72.7, 25.9, 27.0, 23.1, 16.3, 119.3, 140.7, 101.2, 163.8, 101.7, 164.3, 32.9,

CH C CH3 CH3 CH3 CH3 C C C C CH C CH2

8′ 9′ 10′

76.6, CH 21.1, CH3 172.3, C

2.21, 2.03, 1.65, 1.31, 3.40,

overlap overlap m m dd (10.4, 1.7)

1.54, 2.02, 1.90, 1.86, 3.78,

m overlap m m t (7.6)

1.10, 1.18, 1.11, 1.76,

s s s s

30.8, CH2 76.9, CH 86.7, C 34.9, CH2 27.7, CH2

6.27, s 3.03, 2.69, 4.61, 1.47,

123.9, CH 136.0, C 37.4, CH2

dd (16.6, 3.3) dd (16.6, 11.4) overlap d (6.6)

water, pH 7.0). The flasks were incubated at room temperature under static conditions for 35 days. Extraction and Isolation. The cultures (45 L) were filtered through cheesecloth to separate the mycelial mass from the aqueous layer. The filtrate was then extracted three times by shaking with 3-fold volumes of EtOAc, while the mycelium was extracted by acetone. After removing acetone by evaporation under vacuum, the obtained aqueous acetone solution was extracted three times with equal volumes of EtOAc. The combined EtOAc extracts were dried under vacuum to produce 32.1 g of extract. The EtOAc extract was subjected to a silica gel VLC column, eluting with a stepwise gradient of 0%, 10%, 15%, 20%, 40%, 60%, 80%, and 100% EtOAc in petroleum ether (v/v), to give 14 fractions (Fr. 1−14). Fraction 3 (0.6 g) was subjected to HPLC over a πNAP column (80% MeOH−H2O, v/v) to give compounds 12 (tR 8.4 min; 9.6 mg) and 10 (tR 11.8 min; 7.5 mg). Fraction 4 (1.4 g) was applied to octadecylsilane (ODS) silica gel with gradient elution of MeOH−H2O (1:5, 2:3, 3:2, 4:1, 1:0) to yield five subfractions (Fr. 4-1−Fr. 4-5). Fr. 4-3 (51 mg) was subjected to HPLC over ODS (85% MeOH−H2O, v/v) to give compound 7 (tR 7.1 min; 6.8 mg). Fr. 4-5 (45 mg) was subjected to HPLC over a πNAP column (90% MeOH−H2O, v/v) to give compounds 1 (tR 6.9 min; 3.8 mg) and 2 (tR 8.4 min; 4.1 mg). Fraction 5 (1.3 g) was applied to ODS silica gel with gradient elution of MeOH−H2O (1:5, 2:3, 3:2, 4:1, 1:0) to yield four subfractions (Fr. 5-1−Fr. 5-5). Fr. 5-2 (51 mg) was chromatographed on a silica gel column with elution of petroleum ether−EtOAc (5:1) to give compound 4 (7.4 mg). Fr. 5-4 (41 mg) was chromatographed on a silica gel column with elution of petroleum ether−EtOAc (5:1) to give compound 8 (11.0 mg). Fr. 5-3 (21 mg) was subjected to HPLC over ODS (70% MeOH−H2O, v/v) to give compounds 5 (tR 8.2 min; 2.1 mg) and 13 (tR 10.3 min; 6.4

88.1, 72.3, 25.1, 26.3, 22.8, 16.3, 119.4, 140.7, 101.2, 163.8, 101.8, 164.4, 32.9,

CH C CH3 CH3 CH3 CH3 C C C C CH C CH2

76.6, CH 21.1, CH3 172.3, C

2.21, 2.03, 1.71, 1.32, 3.36,

overlap overlap m m dd (10.0, 1.5)

1.59, m 2.00, overlap 1.76, m 1.79, m 3.70 dd (9.4, 6.2) 1.12, 1.14, 1.10, 1.76,

s s s s

6.26, s 3.03, 2.69, 4.61, 1.47,

dd (16.6, 3.3) dd (16.6, 11.4) overlap d (6.6)

mg). Fraction 6 (0.5 g) was subjected to HPLC over ODS (65% MeOH−H2O, v/v) to yield 11 (tR 10.2 min, 12.0 mg) and 9 (tR 14.1 min, 14.2 mg). Fraction 7 (1.3 g) was subjected to Sephadex LH-20 chromatography with CHCl3−MeOH (1:1) to afford five subfractions (Fr. 7-1−Fr. 7-5). Fr. 7-3 (31 mg) was subjected to HPLC over a πNAP column (75% MeOH−H2O, v/v) to give compound 6 (tR 14.7 min; 2.4 mg). Fr. 7-4 (19 mg) was subjected to HPLC over a πNAP column (75% MeOH−H2O, v/v) to give compound 3 (tR 16.8 min; 5.6 mg). Chrodrimanin K (1): colorless crystals; mp 319 °C; [α]25D −41 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (4.59), 277 (4.20), 311 (3.89) nm; ECD (0.18 mM, MeOH) λmax 275 (−8.60), 250 (−1.31), 210 (−6.91) nm; IR (KBr) νmax (cm−1) 3500, 1698, 1663, 1477, 1368, 1150; 1H and 13C NMR data, Table 1; HRESIMS m/z 485.1707 [M + Na]+ (calcd for C25H31ClO6Na, 485.1701). Chrodrimanin L (2): colorless crystals; mp 314 °C; [α]25D −14 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 227 (4.44), 277 (3.99), 312 (3.70) nm; ECD (0.19 mM, MeOH) λmax 273 (−8.14), 250 (−2.83), 237 (−5.15), 210 (−3.52) nm; IR (KBr) νmax (cm−1) 3498, 1702, 1660, 1512, 1362, 1146; 1H and 13C NMR data, Table 1; HRESIMS m/z 519.1310 [M + Na]+ (calcd for C25H30Cl2O6Na, 519.1312). Chrodrimanin M (3): colorless crystals; mp 211 °C; [α]25D −36 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 219 (4.61), 274 (4.12), 312 (3.87) nm; ECD (0.32 mM, MeOH) λmax 274 (−8.55), 238 (−5.58), 210 (−7.06) nm; IR (KBr) νmax (cm−1) 3498, 1648, 1521, 1341, 1130; 1 H and 13C NMR data, Table 1; HRESIMS m/z 469.2191 [M + Na]+ (calcd for C25H34O7Na, 469.2197). Chrodrimanin N (4): colorless crystals; mp 309−310 °C; [α]25D −29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (4.48), 273 (3.98), 312 (3.65) nm; ECD (0.35 mM, MeOH) λmax 273 (−7.73), 237 1045

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(−2.88), 219 (+1.62) nm; IR (KBr) νmax (cm−1) 3508, 1742, 1675, 1477, 1314, 1120; 1H and 13C NMR data, Table 1; HRESIMS m/z 511.2301 [M + Na]+ (calcd for C27H36O8Na, 511.2302). Verruculide B2 (5): colorless oil; [α]25D −42 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.47), 271 (4.15), 310 (3.85) nm; ECD (0.43 mM, MeOH) λmax 273 (−3.71), 237 (−6.92), 219 (−5.52) nm; IR (KBr) νmax (cm−1) 3445, 1649, 1548, 1430, 1375, 1260; 1H and 13C NMR data, Table 2; HRESIMS m/z 471.2350 [M + Na]+ (calcd for C25H36O7Na, 471.2353). Verruculide B3 (6): colorless oil; [α]25D −45 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 218 (4.52), 271 (4.16), 311 (3.86) nm; ECD (0.27 mM, MeOH) λmax 273 (−3.19), 239 (−5.29), 215 (−3.19) nm; IR (KBr) νmax (cm−1) 3447, 1654, 1542, 1429, 1364, 1267; 1H and 13C NMR data, Table 2; HRESIMS m/z 471.2354 [M + Na]+ (calcd for C25H36O7Na, 471.2353). X-ray Crystal Data for 1−4, 9, and 11. Colorless crystals of 1−4, 9, and 11 were obtained in the mixed solvent of MeOH and H2O. Crystal data of 1−4 and 11 were obtained on a Bruker D8 QUEST diffractometer (Bruker) with graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å). Crystal data of 9 were obtained on a Bruker Smart APEX II area detector diffractometer with graphitemonochromated Mo Kα radiation (λ = 0.710 73 Å). Crystallographic data for 1−4, 9, and 11 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication numbers CCDC 1517211, 1517208, 1517210, 1517207, 1517221, and 1517209, respectively. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Crystal data for 1: orthorhombic, C25H31ClO6; space group P 21 21 21 with a = 15.2749(18) Å, b = 15.1875(17) Å, c = 10.1806(12) Å, V = 2361.8(5) Å3, Z = 4, Dcalcd = 1.302 g/cm3, μ = 1.750 mm−1, and F(000) = 984. T = 273(2) K. R1 = 0.0649 (I > 2σ(I)), wR2 = 0.1526 (all data), S = 1.058. Absolute structure parameter: 0.06(3). The structures were solved using ShelXS. The structural solutions were found by direct methods and refined using the ShelXL package by least-squares minimization. The final structures were examined using the Addsym subroutine of PLATON to ensure that no additional symmetry could be applied to the models. All non-hydrogen atoms were refined with anisotropic thermal factors. Crystal data for 2: orthorhombic, C25H30Cl2O6; space group P 21 21 21 with a = 11.0269(3) Å, b = 13.3801(3) Å, c = 16.1400(4) Å, V = 2381.31(10) Å3, Z = 4, Dcalcd = 1.387 Mg/m3, μ = 2.784 mm−1, and F(000) = 1048. T = 273(2) K. Absolute structure parameter: 0.042(11). The structure was solved by direct methods (ShelXL) and expanded using Fourier techniques (ShelXL). The final cycle of fullmatrix least-squares refinement was based on 4332 unique reflections (2θ < 50°) and 305 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0575, wR2 = 0.1103, and R = 0.0786 for I > 2σ(I) data. Crystal data for 3: monoclinic, C25H34O7; space group P 21 with a = 19.6602(15) Å, b = 14.7796(12) Å, c = 26.1250(19) Å, α = γ = 90°, β = 91.529(4)°, V = 7588.4(10) Å3, Z = 2, Dcalcd = 1.208 Mg/m3, μ = 0.736 mm−1, and F(000) = 2960. T = 273(2) K. Absolute structure parameter: 0.02(1). The structure was solved by direct methods (ShelXL) and expanded using Fourier techniques (ShelXL). The final cycle of full-matrix least-squares refinement was based on 25 280 unique reflections (2θ < 50°) and 1829 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0988, wR2 = 0.2565, and R = 0.1573 for I > 2σ(I) data. Crystal data for 4: orthorhombic, C27H36O8; space group P 21 21 21 with a = 10.6191(4) Å, b = 13.3554(5) Å, c = 17.1996(6) Å, V = 2439.29(15) Å3, Z = 4, Dcalcd = 1.330 Mg/m3, μ = 0.801 mm−1, and F(000) = 1048. T = 273(2) K. Absolute structure parameter: 0.12(18). The structure was solved by direct methods (ShelXL) and expanded using Fourier techniques (ShelXL). The final cycle of full-matrix leastsquares refinement was based on 4284 unique reflections (2θ < 50°) and 324 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0764, wR2 = 0.1666, and R = 0.1201 for I > 2σ(I) data.

Crystal data for 9: orthorhombic, C25H34O6·H2O; mp 304 °C; space group P 21 21 21 with a = 11.144(8) Å, b = 16.201(12) Å, c = 12.443(10) Å, V = 2247(3) Å3, Z = 4, Dcalcd = 1.326 Mg/m3, μ = 0.096 mm−1, and F(000) = 968. T = 296(2) K. The structure was solved by direct methods (ShelXL) and expanded using Fourier techniques (ShelXL). The final cycle of full-matrix least-squares refinement was based on 5696 unique reflections (2θ < 50°) and 300 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0695, wR2 = 0.1364, and R = 0.2116 for I > 2σ(I) data. Crystal data for 11: orthorhombic, C25H30O7; mp 343 °C; space group P 21 21 21 with a = 10.9888(4) Å, b = 12.7811(4) Å, c = 15.6040(5) Å, V = 2191.57(13) Å3, Z = 4, Dcalcd = 1.341 Mg/m3, μ = 0.802 mm−1, and F(000) = 944. T = 273(2) K. Absolute structure parameter: 0.04(8). The structure was solved by direct methods (ShelXL) and expanded using Fourier techniques (ShelXL). The final cycle of full-matrix least-squares refinement was based on 3976 unique reflections (2θ < 50°) and 294 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0415, wR2 = 0.1028, and R = 0.0450 for I > 2σ(I) data.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01061. HRESIMS, IR, UV, and 2D NMR spectra of compounds 1−6, 18S rRNA gene sequences of Penicillium sp. SCSKFD09, and quantum calculation details (PDF) Crystallographic data (CIF) (CIF) (CIF) (CIF) (CIF) (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax (H.-F. Dai): +86-898-66961869. E-mail: daihaofu@ itbb.org.cn. *Tel/Fax (Y.-X. Zhao): +86-898-66989095. E-mail: [email protected]. ORCID

You-Xing Zhao: 0000-0002-8107-2510 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (41606088), Special Fund for Agro-Scientific Research in the Public Interest (201303117), and Fundamental Scientific Research Funds for CATAS (ITBB2017).

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REFERENCES

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